High-resolution HIV-1 m⁶A epitranscriptome reveals isoform-dependent methylation clusters and unique 2-LTR transcript...

The N⁶ -methyladenosine (m⁶A) modification of HIV-1 has been widely studied but the number and precise positions of the m⁶A sites remain unclear due to the lack of precision of detection methods. Using the latest Nanopore chemistry and direct m⁶A base-calling, we identified 18 m⁶A: 14 at the 3' end and 4 in central regions of the genome. Our data reveal differential methylation of these positions between splicing isoforms. Eleven of these sites are clustered in two short segments with peak-shaped methylation profiles. Single-molecule analysis revealed that a very small number of transcripts were unmethylated in both clusters. We also identified a ∼732 nt RNA species resulting from the transcription of non-integrated viral DNA circles closed by two long terminal repeats. These transcripts started in the first LTR, terminated at the polyA site of the second LTR, and harbored six m⁶A sites. Five of these sites were present in other transcripts and, remarkably, had the highest methylation rates. The sixth site was methylated only in this transcript, suggesting a role for this RNA in HIV-1 infection. These findings reveal a new landscape of HIV m⁶A transcriptome modifications and pave the way for studies deciphering their role in the viral life cycle.